This invention concerns acyclic nucleotide analogs, their preparation and use. In particular it concerns separate enantiomers of 2-phosphonomethoxypropyl derivatives of purine and pyrimidine bases.
There is an urgent need for development of chemotherapeutic agents in the therapy of viral diseases. In particular treatment of diseases caused by retroviruses presents one of the most difficult challenges in human medicine. While a number of antiviral agents which are registered or are currently under study can effectively cure disease, relieve symptoms or substantially prolong the intervals among the recurrences of certain chronic viral infections, such positive outcomes have not yet been achieved in many instances, notably that of AIDS, as an example of retroviral disease. Selectivity of antiviral action, which is an important requirement for novel antiviral agents, has not been achieved.
Most of the compounds which are clinically useful for antiviral chemotherapy are nucleosides, modified in either the purine or pyrimidine base and/or the carbohydrate moiety. Such compounds mainly act in processes related to the synthesis of viral nucleic acids; their action depends on ability to undergo phosphorylation and subsequent transformation to the triphosphates. One problem in administering modified nucleosides is the absence of suitable phosphorylating activity in the host cell and the existence of viral strains lacking virus-specific phosphorylating activity. While enzymatically resistant nucleotide analogs might appear to be particularly useful as potential antivirals, their polar character prevents effective entry of these analogs into the cells, as does lack of appropriate nucleotide receptors at the cellular membrane.
This difficulty appears to be overcome in the series of acyclic nucleotide analogs which contain an aliphatic chain, bearing hydroxyl groups, replacing the sugar moiety. For example, the phosphates or phosphonic acid derivatives derived from the antiviral nucleoside analog ganciclovir (Cytovene) are reported to possess an anti-herpes virus activity (Reist at al., in xe2x80x9cNucleotide Analogs as Antiviral Agentsxe2x80x9d, ACS Symposium Series, No. 01, pp. 17-34 (1989); Tolman, ibid, pp. 35-50; Prisbe et al., J Med Chem (1986), 29:671).
The following formulas describe several classes of prior art compounds: 
Another group of antiviral compounds where the antiviral action is less strictly limited by the nature of the heterocyclic base includes phosphonic acid analogs in which a phosphonic acid moiety is linked to the hydroxyl group of an aliphatic chain sugar substitute via a methylene unit. Examples of such compounds are HPMP-derivatives (1) which were disclosed by the UK Patent Application No. 2 134 907 and PV-3017, now published on 30 Dec. 1986 as EP 206,459 of Holÿ et al. Such compounds act exclusively against DNA viruses as reported by De Clercq et al. in Nature (1986) 323:464-467, and reviewed by Holÿ et al. in Antiviral Res (1990) 13:295.
A similar type of antivirals is represented by PME-derivatives (2) disclosed by European Patent Application 0 206 459 by Holÿ et al. and described in detail by De Clercq at al. in Antiviral Res (1987) 8:261, and by Holÿ et al. in Collection Czech Chem Commun (1987) 52:2801; ibid. (1989), 54:2190). These compounds act against both DNA viruses and retroviruses, including HIV-1 and HIV-2. The adenine derivative, PMEA, was demonstrated to exhibit an outstanding activity against Moloney sarcoma virus in mice, simian immunodeficiency virus in Rhesus monkeys as well as feline immunodeficiency virus in cats (Balzarini et al., AIDS (1991) 5:21; Egberink et al., Proc Natl Acad Sci U.S.A. (1990) 87:3087).
The extensive structure-activity investigation which concentrated on the modification of the side-chain (described by Holÿ et al. in xe2x80x9cNucleotide Analogs as Antiviral Agentsxe2x80x9d, ACS Symposium Series No.401 (1989), p. 51) did not reveal any additional substantially active antivirals. Replacement of this hydroxyl by fluorine atom resulted in the FPMP-compounds (3) which, in addition to having some anti-DNA-virus activity display a substantial effect on both HIV-1, HIV-2 and murine sarcoma virus (as taught by Holÿ et al., Czechoslovak Patent Application PV 2047-90 now published on Oct. 30, 1991 as EP 454,427, and by Balzarini et al., Proc Natl Acad Sci U.S.A. (1991) 88:4961).
The racemic mixtures of 9-(2-phosphono-methoxypropyl) adenine and guanine (PMPA and PMPG) were also described by Holÿ et al., European Patent Appl. 0206459 (PMPA) and Holÿ et al., Collection Czech Chem Commun (1988) 53:2753 (PMPA), and by U.S. patent. application Ser. No. 932,112 (PMPG). PMPA was devoid of any appreciable antiherpetic effect while any antiherpetic activity of PMPG appeared due to its substantial cytotoxicity. The clinical forms of PMPG, and of the related compound, 9-C3-hydroxy-2-(phosphonomethoxy)propyl) guanine (HPMPG) are disclosed in EP application 452935. For these guanine forms, the R-enantiomers consistently gave greater antiviral activity, especially in regard to the retrovirus HIV. There was little difference between RandS enantiomers in antiviral activity with regarding to some DNA viruses. It cannot be predicted whether this pattern of activity would extend to PMP compounds of other than guanine.
Nothing in the above-cited references or their combination permits any prediction that the resolved enantiomers of the present invention would exhibit antiretroviral activity, or what the enantiomers preference would be.
Resolved enantiomeric forms of N-(2-phosphono-methoxypropyl) derivatives of purine and pyrimidine bases have been synthesized and found to possess useful and unexpected antiviral activity which is directed specifically against retroviruses. These compounds are of the formulas IA and IB, wherein IA represents the R enantiomer and IB represents the S enantiomer. 
In the formulas IA and IB, B is a purine or pyrimidine base or aza and/or deaza analog thereof.
Thus, in one aspect, the invention is directed to compositions comprising Formula IA unaccompanied by any substantial amount of the corresponding compound of Formula IB and to compositions comprising a compound of the Formula IB unaccompanied by any substantial amount of the corresponding compound of the formula IA.
By xe2x80x9cany substantial amountxe2x80x9d is meant less than about 5 mole %, preferably less than about 2 mole %, more preferably less than about 1 mole % and most preferably in undetectable amounts. By xe2x80x9ccorresponding compoundxe2x80x9d is meant the enantiomer of the compound shown.
Other aspects of the invention include the preparation of these compositions, their formulation into antiviral pharmaceutical compositions and the use of these formulations to treat retroviral infections.
The compounds of the invention are the resolved (R) and (S)-enantiomers of N-(2-phosphonomethoxypropyl) derivatives of purine and pyrimidine bases which have structural formula I. 
B is a purine or pyrimidine base or an aza and/or deaza analog thereof other than guanine. Renantiames are preferred. As used herein, xe2x80x9cpurinexe2x80x9d refers to substituted or unsubstituted moieties of the formula: 
and xe2x80x9cpyrimidinesxe2x80x9d to substituted or unsubstituted moieties of the formula 
In aza analogs, at least one C shown in the above formulas is replaced by N; in deaza analogs, at least one N is replaced by C. Combinations of such replacements are also included within the scope of the invention.
Thus, 3-deaza purine analogs are of the formula 
8-aza purine analogs are of the formula 
and 1-deaza-8-aza purine analogs are of the formula 
Preferred embodiments of B are those wherein B is a purine base selected from the group consisting of adenine, 2, 6-diaminopurine, 2-aminopurine, hypoxanthine, xanthine; and their 1-deaza, 3-deaza or 8-aza analogs;
derivatives of purine or said analogs, which derivatives are other than guanine, substituted at position 2 and/or 6 and/or 8 by amino, halogen, hydroxy, alkoxy, alkylamino, dialkylamino, aralkylamino, heteroaralkylamino, hydroxyamino, alkoxyamino, hydrazino, azido, mercapto or alkylthio.
Also preferred are those embodiments wherein B is a pyrimidine base selected from the group consisting of cytosine, uracil, thymine, 5-methylcytosine, and their 6-aza analogs; and
derivatives of pyrimidine substituted at the exocyclic amino group in position 4 by alkyl, aralkyl, hydroxy or amino.
As used herein, halogen refers to F, Cl, Br or I; alkyl refers to a straight- or branched-chain saturated hydrocarbyl group containing 1-6C, such as methyl, ethyl, isopropyl, n-pentyl, neopentyl and the like; alkoxy is a group of the formula xe2x80x94OR wherein R is alkyl as above defined; alkylthio is a group of the formula xe2x80x94SR wherein R is alkyl as above defined; aralkyl or heteroaralkyl is a group of the formula xe2x80x94Rxe2x80x94Ar wherein xe2x80x94Rxe2x80x94 is the alkylene counterpart of alkyl (xe2x80x94R) as above defined and Ar is a substituted or unsubstituted aromatic group of 6-10C including phenyl, napthyl, quinolyl and the like. Particularly preferred is benzyl.
The compounds of the invention can be isolated in the form of free acids, salts or, in the case of compounds with heterocyclic bases bearing at least one amino function, in the form of zwitterions. The acid or zwitterionic forms can be obtained on purification of the deionized crude material by anion exchange chromatography, using volatile organic acids (acetic or formic acid) as eluents. The free acid forms can be easily transformed into physiologically acceptable salts by methods known in the art. Such salts include those of ammonium ion, Li+, Na+, K+, Mg++ and Ca++ or pharmaceutically acceptable organic cations; the salts may be monobasic or dibasic. Compounds with at least one amino function contained in B can also be prepared as the acid addition salts with inorganic or organic acids, such as HBr, HCl, H2SO4 or HOAc.
In certain cases, the acid or zwitterionic forms of compounds of the Formula IA and IB are extremely water-insoluble. Under such circumstances, purification is performed on a medium basic anion exchanger (e.g., DEAE-cellulose, DEAE-Sephadex) in a weakly alkaline volatile buffer, such as triethylammonium hydrogen carbonate. The resulting water-soluble triethylammonium salts can be transformed to salts of other cations by, e.g., cation exchange, using cation exchanger in the corresponding form.
The free acids, zwitterions or salts of compounds of Formula IA or IB are stable in the solid state or in sterile aqueous or aqueous-alcoholic solutions.
The compounds of this invention can be prepared from an easily prepared chiral intermediate X derived from resolved lactic acid ester enantiomers using reaction scheme 1 or 2.
Reaction scheme 1 is as follows: 
wherein B is as defined above and Bxe2x80x2 is its suitably protected form; the * above the chiral center indicates that the resolved enantiomer is used. Protection of B comprises blocking of active hydrogen atoms contained in B, such as any hydroxy, amino or carbamido group. Protection can be achieved by introduction of alkali-labile groups, such as acetyl, benzoyl, pivaloyl or amidine, such as a dimethylaminomethylene group, or, by acid-labile groups such as trityl, substituted trityl, tetrahydropyranyl groups and the like.
The desired enantiomer of 2-0-tetrahydro-pyranylpropane-1,2-diol of the Formula X is transformed to the corresponding 1-0-p-toluenesulfonyl esters in Step 1 under usual conditions, i.e. reaction with p-toluenesulfonyl chloride in pyridine. The tosyl group shown is preferred, however standard organic leaving groups such as mesylate, triflate or halide can also be used.
The protected intermediate of the Formula XI, isolated either by direct crystallization or by silica gel chromatography, occurs as a mixture of diastereomers which gives complex NMR spectra.
The alkylation of a heterocyclic base with the synthon of the Formula XI in Step 2 is mediated by the formation of an anion which can be generated either by pretreatment of the base with alkali hydride in an inert solvent, such as dimethylformamide, or, by the anion formation generated in situ by alkali carbonate. In the latter case, an importance of cesium carbonate as a catalyst must be recognized. This catalyst not only substantially accelerates the alkylation of the base, it also favorably influences the regiospecificity of alkylation by the synthon XI in purine ring systems, giving alkylation at the preferred N9 position of purines or the corresponding position in the aza or deaza bases.
The tetrahydropyran-2-yl group is cleaved in acidic media to afford the intermediate III in Step 3. This cleavage can be achieved by the action of mineral acid (e.g. sulfuric acid) anion exchange resins or organic acids (e.g. acetic acid, formic acid) followed by deionization.
The N-(2-hydroxypropyl) derivatives of Formula III are transformed in Step 4 to base-protected derivatives of Formula V using any of a variety of methods generally available for the purpose, such as selective N-acetylation, N-benzylation, reaction with dimethyformamide dialkyl acetals, N-tritylation, reaction with 3,4-dihydro-2H-pyrane and the like.
In Step 5 of Scheme 1 the protected intermediate of Formula V is converted to the alkoxide anion by treatment with a suitable base, such as alkali metal hydride, in a non-reactive solvent, such as dimethylformamide, and the alkoxide is treated with dialkyl p-toluenesulfonyloxymethylphosphonate (Formula IV). Preferably the phosphonate esters are of isopropyl alcohol. The reaction is performed by stirring a mixture of the Formula V intermediate with the tosyl derivative of Formula IV in the presence of three equivalents (relative to intermediate V) of alkali hydride, e.g., NaH or KH or other suitable reagents at temperatures ranging from xe2x80x9410xc2x0 C. to 80xc2x0 C., mostly from 0xc2x0 C. to 20xc2x0 C. The reaction lasts from several hours up to several days, depending on the nature and concentration of the reaction components. Since gaseous hydrogen is evolved during the reaction, it is essential to work in an open system with suitable protection against moisture.
Protecting groups are then removed from Bxe2x80x2 and the phosphonate ester linkages are hydrolyzed. Removal of the protecting groups from Bxe2x80x2 in Step 6 can be achieved by generally acceptable methods such as methanolysis, acid hydrolysis, etc. Alkali labile groups can be removed simply by dilution of the mixture with methanol. The resulting diester of Formula VII is isolated by silica gel chromatography, or using other suitable supports, or contaminating non-nucleotide materials may be removed by deionization on cation exchangers, such as Dowex 50, or, by hydrophobic silica chromatography. The purified intermediate of Formula VII is then hydrolyzed, in Step 7 for example, by treating with a halotrialkylsilane, such as bromotrimethylsilane or iodotrimethylsilane in a polar aprotic solvent such as acetonitrile or DMF for 4-20 hours at room temperature. Volatiles are then evaporated in vacuo and the final product may then be obtained by further purification and isolation techniques depending upon its character. Ion exchange chromatography making use of the presence of negatively charged phosphonate group is preferred.
Alternatively, compounds of Formula I can be prepared by Reaction Scheme 2. 
In Scheme 2, the synthon of Formula XVII is ultimately used to provide the chiral PMP precursor. It bears a leaving tosyl group and can be used for alkylation of the heterocyclic base or its protected derivative to afford Formula VII, the protected diester form of the compounds of the invention.
In Reaction Scheme 2, as in Reaction Scheme 1, a resolved form of 2-0-(tetrahydropyranyl)-propane-1,2-diol of the Formula X provides the required resolved enantiomer. The resolved compound of Formula X affords, in Step 1, on benzylation under standard conditions, e.g. with benzyl bromide in the presence of sodium hydride in DMF, the benzyl ether XII which is then transformed by acid hydrolysis in Step 2 to the resolved enantiomeric 2-0-benzylpropane-1,2-diol of the Formula XIII. Either enantiomer (a distillable oil) affords, in Step 3, on chloromethylation with 1,3,5-trioxane or paraformaldehyde in the presence of hydrogen chloride an intermediary chloromethyl ether of the Formula XIV which is, without purification, transformed in Step 4 into the phosphonate diester XV by heating with tri(2-propyl)phosphite with simultaneous removal of 2-propyl chloride. Though the intermediate of the Formula XV is distillable in a high vacuum, this procedure may result in racemization. Partially purified products of this reaction are then hydrogenolysed under standard conditions such as hydrogenation in the presence of palladium-on-charcoal catalyst in methanol and the intermediary diester of the Formula XVI resulting from Step 5 is, without isolation, transformed in Step 6 into the tosyl derivative XVII by the action of tosyl chloride in pyridine.
The sequence Xxe2x86x92XVII involves six steps, but does not require purification of the intermediates. All reactions proceed with high conversion so that the over-all yield of the sequence exceeds 40%. 2-propyl esters of the phosphonate are preferred, but other phosphonate protecting ester groups such as methyl, ethyl, benzyl and cyclic diesters can be used to the same effect. Also the tosyl group in the synthon of the Formula XVII could be replaced by other leaving groups, as for example mesyl, triflyl, p-nitrophenylsulfonyl, etc.
Step 7 of this synthetic sequence consists in the alkylation of the heterocyclic base by the synthon of the Formula XVII. It requires an equimolar amount of the base relative to the heterocycle. The alkylation is best performed in DMF at increased temperature with either a sodium salt generated from the heterocyclic base by sodium hydride reaction or, alternatively, with a mixture of the heterocyclic base and a slight excess of potassium carbonate or, to an advantage, cesium carbonate. The reaction can be made either with unprotected or protected (e.g. N-benzoylated) bases or their precursors as mentioned in the description of the reaction according to the Scheme 1.
The alkylation proceeds rapidly and the required intermediate of the Formula VII can be easily isolated from the reaction mixture and purified by chromatography. Further processing of the protected intermediate leading to the compounds of the Formula IA and IB is identical with the procedure described in the Scheme 1.
An advantage of this method of preparation of the Formula I compounds over the method described by the Scheme 1 consists, in addition to the possible avoidance of base protection, in the elimination of acidic conditions which are essential for the preparation of the intermediary N-(2-hydroxypropyl) derivatives of the Formula II, as well as of any other deprotection except for ultimate halotrimethylsilane treatment. The alternative procedure can thus be applied for the syntheses of compounds of the Formula I bearing sensitive heterocyclic bases.
In respect to both reaction Schemes 1 and 2, the compounds of the invention may be prepared by alkylation of the desired heterocyclic base B as shown, or, in certain cases, by alkylation of a precursor of B. Thus, guanine derivatives can be best synthesized via alkylation of 2-amino-6-chloropurine followed by acid hydrolysis of the Cxe2x80x94Cl linkage. Cytosine derivatives can be synthesized by direct alkylation of cytosine in the presence of cesium carbonate in a modest yield; better yields are obtained by the ammonolysis of an intermediate formed by alkylation of 4-methoxy-2-pyrimidone with synthon XI.
Similar subsequent changes at the heterocyclic base can be performed with the final products of the Scheme, i.e. the compounds of Formula I which are the subjects of the invention: adenine, 2,6-diaminopurine or guanine derivatives can be transformed by deamination with nitrous acid or its esters to the corresponding hypoxanthine, 2-hydroxyadenine or xanthine derivatives; similarly, uracil derivatives can similarly be converted to cytosine derivatives. Further transformations of the compounds of the Formula I can be realized with routine methods of nucleic acid chemistry: e.g., bromination of purine base to obtain the 8-bromo derivatives, N-alkylation of the NH-functions in both the purine and pyrimidine compounds, etc. None of these subsequent transformations concerns any changes at the PMP group of the compounds of Formula I.
It will be recognized that the intermediate compounds that are parts of the pathways of Schemes 1 and 2 are themselves novel compounds and therefore are part of the invention.
An advantage of using the processes of Schemes 1 and 2 consists in the utilization of starting materials of the Formula X, which are easily available in optically pure forms. The reaction sequence to prepare the chiral synthon X used both in Scheme 1 and Scheme 2 is described in Scheme 3. 
The crucial step of every asymmetric synthesis depends on the availability of optically pure chiral starting materials. The method used in the present invention makes use of commercially available (Merck) enantiomers of lactic acid alkyl esters of Formula VIII. These esters are first protected at the hydroxyl function by tetrahydropyranyl group; this reaction is performed without solvent by direct addition in the presence of an acid catalyst. The esters of the Formula IX are obtained by fractionation in vacuo. These intermediates are reduced by lithium aluminum hydride in ether or by bis(2-methoxyethoxy)aluminum hydride in ether or other inert solvents to the compounds of Formula X.
The enantiomerically resolved compounds of the invention display significant antiretroviral activity both in vitro and in vivo. Their in vitro efficacy was demonstrated on human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2) in MT4 and CEM cells, as well as on Moloney murine sarcoma virus (MSV) in C3H/3T3 cells. Their in vivo efficacy was proven in MSV infected NMRI mice, where the compounds markedly postponed the mean day of tumor initiation and substantially prolonged the mean survival day, both upon parenteral and oral administration.
The compounds of the invention have several advantages over the prototype compounds (PMEA, PMEADAP, PMEG) and/or FPMP derivatives, e.g., FPMPA, FPMP-DAP), and most relevant, unresolved PMPG +PMPA: (a) their antiretroviral activity is clearly separated from other antiviral activities (e.g. against herpes viruses); (b) their antiretroviral activity is not due to cellular toxicity. Consequently, the in vitro. therapeutic index of these compounds is much higher than those of the prototype compounds and reaches a value of  greater than 2000 in some of the cases. Such compounds can be ideally suited to a long-term treatment of chronic diseases, e. g. AIDS.
The structures of the test compounds presenting the subject of this invention are completely unrelated to any of the compounds currently used in clinical trials of AIDS patients. This will avoid cross-resistance of these compounds against those virus strains that became resistent to such treatment, e.g., AZT, DDI, DDC, TIBO, nevirapine, pyridinone, etc.
The biological activity of these compounds is highly enantiospecific. Generally, the (R)-enantiomers are responsible for the antiretroviral activity. The activity of (S)-enantiomers in the guanine series is accompanied by a substantially increased toxicity.
The compounds of the invention can be applied for the treatment of diseases caused by retroviruses, e.g. human immunodeficiency viruses (AIDS), human T-cell leukemia virus (hairy cell leukemia, acute T-cell leukemia (HTL)). Since the molecular target of antiviral action of these compounds is the virus-encoded reverse transcriptase, they should also have anti-hepadna virus activity (e.g. hepatitis B virus).
The compounds may be administered topically or systemically i.e. orally, rectally, intravaginally and parenterally (by intermuscular, intravenous, subcutaneous and nasal routes). Generally, the oral application will require a larger quantity of the active ingredient to produce a therapeutic effect comparable with quantity given parenterally.
Pharmaceutical compositions for the treatment of human retroviral diseases will comprise at least one compound of the Formula IA or IB or a pharmaceutically acceptable salt thereof, generally comprising 95 to 0.5% wt/wt of the composition in combination with a pharmaceutically acceptable carrier and non-toxic inert adjuvant. Other therapeutic agents can also be present. Additionally, mixtures of compounds of formulas IA and/or IB can be employed, provided that each member of such mixture is substantially free of its enantiomer.
Pharmaceutical compositions containing compounds of the Formula I are conventionally prepared as tablets, lozenges, capsules, powders, aqueous or oil suspensions, syrups and aqueous solutions. The compounds can be formulated for a variety of modes of administration including systematic, topical or localized administration. Techniques and formulations generally may be found in Remington""s Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., latest edition. The active ingredient is generally combined with a carrier such as a diluent or excipient which may include fillers, extenders, binders, wetting agents, disintegrants, surface-active agents, or lubricants, depending on the nature of the mode of administration and dosage forms. Typical dosage forms include tablets, powders, liquid preparations including suspensions, emulsions and solutions, granules, capsuels and suppositories, as well as liquid preparations for injections, including liposome preparations.
For systematic administration, injection is preferred, including transmuscular, intravenous, intraperitoneal, and subcutaneous. For injection, the compounds of the invention are formulated in liquid solutions, preferably in physiologicallay compatible buffers such as Hank""s solution or Ringer""s solution. In addition, the compounds may be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms are also included. Systematic administration can also be by transmucosal or transdermal means, or the compounds can be administered orally. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, bile salts and fusidic acid derivatives for transmucosal administration. In addition, detergents may be used to facilitate permeation. Transmucosal administration may be through use of nasal sprays, for example, or suppositories. For oral administration, the compounds are formulated into conventional oral administration forms such as capsules, tablets, and tonics.
For topical administration, the compounds of the invention are formulated into ointments, salves, gels, or creams, as is generally known in the art. The compounds may also be administered for ophthalmic or ocular indications when appropriately formulated.
The effective dose of active compound of the present invention is expected to be about 0.01-50 mg/kg body weight with preferred range of 1 to 20 mg/kg. The dosage in clinical applications must be professionally adjusted considering age, weight and condition of the patient, the route of administration, the nature and gravity of the illness. Generally, the preparations are expected to be administered by oral route from about 100 mg to about 1000 mg per day, one to three times a day.
The compounds of the invention, their methods of preparation and their biological activity will appear more clearly from the examination of the following examples which are presented as an illustration only and are not to be considered as limiting the invention in its scope. All melting points have been estimated with the use of Kofler""s block and are uncorrected. Solutions were evaporated at 40xc2x0 C./2 kPa when not specified. Thin-layer chromatography was made with the use of silica plates containing fluorescent indicator; detection by UV-light. The nuclear magnetic resonance (NMR) spectral characteristics refer to chemical shifts (xcex4) expressed in parts per million (ppm) vs. tetramethylsilane (TMS) as reference compound. The multiplicity of signals is reported as singlet (s), doublet (d), doublet of doublets (dd), multiplet (m), triplet (t) or quartet (q); other abbreviations include broad (br) signal, aromatic protons (arom.), d6-DMSO for hexadeuteriodimethylsulfoxide, D2O for deuterium oxide, NaOD for sodium deuteride and CDCl3 for deuteriochloroform. Other abbreviations are conventional.